GSM6774296: KO Female HFD GEX, scRNA seq; Mus musculus; RNA-Seq
Visceral adipose tissue was dissected from visceral fat pads, rinsed in ice cold 1X PBS and minced with a razor blade. Fat was digested enzymatically with 3 mg/ml collagenase/dispase at 37 C for 1 hour. Suspension was passed through a 70um strainer and centrifuged to pellet stromal vascular fraction. Cell suspension was resuspended in 1X PBS/0.04% BSA and counted. Libraries were prepared according to the manufacturer's instructions (Chromium Next GEM Single Cell 3' v3.1 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Feature barcode for cell multiplexing oligo label, Unique Molecular Identifier (UMI) and the 10x Barcode. For the gene expression and CellPlex library construction, the pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction, Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added to ensure that no sample indices overlapped between different libraries.